Design, expression and characterization of single chain Fv, Mms13 and the single chain Fv-mms13 fusion protein

摘要

Single chain Fv (scFv) antibodies are attractive as tumor-targeting vehicles due to their smaller size compared with intact antibody molecules. Mms13 is a putative membrane anchor protein of magnetosome. The present study fused the scFV gene of type IV collagenase to mms13 using the splicing by overlap extension polymerase chain reaction technique. The genes of scFv, mms13 and the scFv-mms13 fusion gene were cloned into a pET30a(+) vector to construct pET30a(+)-scFv, pET30a(+)-mms13 and pET30a(+)-scFv-mms13 expression vectors. The three protein compositions were confirmed by DNA sequencing and western blot analysis, and their cellular locations were determined using SDS-PAGE. The results of enzyme-linked immunosorbent assays and immunofluorescence demonstrated that the ScFv and ScFv-mms13 fusion proteins bound to the type IV collagenase and the antigen-associated cancer cells SMMC-7721, MCF-7 and HepG2 cells, in a dose-dependent and saturable manner. Although the immunoreactivities of ScFv-mms13 to the type IV collagenase and associated tumor cells were marginally lower than the corresponding scFv (3G11), considerable binding ability to the antigen by ScFv-mms13 remained.